Characterization of a Novel Subtype of Human G Protein-coupled Receptor for Lysophosphatidic Acid

The recent identification of the Vzg-1/Edg2 protein as a functional G protein-coupled receptor for lysophosphatidic acid (LPA) has allowed a sequence-based search for new genes that may encode novel subtypes of LPA receptors. A human cDNA encoding a G protein-coupled receptor, designated Edg4, was identified by searching the GenBank™ for homologs of the human Edg2 LPA receptor. The Edg4 protein is 46% identical and 72% similar in amino acid sequence to human Edg2. When overexpressed in Jurkat T cells, the Edg4 protein mediated LPA-induced activation of a serum response element reporter gene with LPA concentration dependence (EC50 of 10 nm) and specificity. This LPA-induced reporter gene activation could be partially inhibited by pretreatment with pertussis toxin or C3 exoenzyme, suggesting requirements for both a Gi protein and Rho GTPase. Overexpression of Edg4 in Jurkat cells also led to increases in specific binding sites for 3HLPA. Northern blots revealed that twoedg4 mRNA transcripts of 1.8 and 8 kilobases are distributed very differently from edg2 mRNAs in adult human tissues and several cancer cell lines. The existence and distinctive tissue expression of structurally different subtypes of LPA receptors may provide one basis for tissue-specific functions and permit independent regulation of each subtype of LPA receptor. The recent identification of the Vzg-1/Edg2 protein as a functional G protein-coupled receptor for lysophosphatidic acid (LPA) has allowed a sequence-based search for new genes that may encode novel subtypes of LPA receptors. A human cDNA encoding a G protein-coupled receptor, designated Edg4, was identified by searching the GenBank™ for homologs of the human Edg2 LPA receptor. The Edg4 protein is 46% identical and 72% similar in amino acid sequence to human Edg2. When overexpressed in Jurkat T cells, the Edg4 protein mediated LPA-induced activation of a serum response element reporter gene with LPA concentration dependence (EC50 of 10 nm) and specificity. This LPA-induced reporter gene activation could be partially inhibited by pretreatment with pertussis toxin or C3 exoenzyme, suggesting requirements for both a Gi protein and Rho GTPase. Overexpression of Edg4 in Jurkat cells also led to increases in specific binding sites for 3HLPA. Northern blots revealed that twoedg4 mRNA transcripts of 1.8 and 8 kilobases are distributed very differently from edg2 mRNAs in adult human tissues and several cancer cell lines. The existence and distinctive tissue expression of structurally different subtypes of LPA receptors may provide one basis for tissue-specific functions and permit independent regulation of each subtype of LPA receptor. Lysophosphatidic acid (LPA) 1The abbreviations used are: LPA, lysophosphatidic acid (1-acyl-2-hydroxy-sn-glycero-3-phosphate); PA, phosphatidic acid; SRE, serum response element; G protein, guanine nucleotide-binding protein; GPCR, G protein-coupled receptor; PTX, pertussis toxin; kb, kilobase(s). is a lipid mediator with diverse biological activities (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 2Moolenaar W.H. Kranenburg O. Postma F.R. Zondag C.M. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar). LPA is generated by phospholipase cleavage of membrane phospholipids from stimulated cells, especially activated platelets (3Gaits F. Fourcade O. Le Belle F. Gueguen G. Gaige B. Gassama-Diagne A. Fauvel J. Salles J.-P. Mauco G. Simon M.-F. Chap H. FEBS Lett. 1997; 410: 54-58Crossref PubMed Scopus (147) Google Scholar). LPA is present at micromolar concentrations in serum and accounts for much of the cellular proliferative effect of serum (4Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3832) Google Scholar, 5Tigyi G. Miledi R. J. Biol. Chem. 1992; 267: 21360-21367Abstract Full Text PDF PubMed Google Scholar, 6Eichholtz T. Jalink K. Fahrenfort I. Moolenaar W.H. Biochem. J. 1993; 291: 677-680Crossref PubMed Scopus (577) Google Scholar). LPA elicits numerous biological functions in addition to proliferation, such as platelet aggregation, smooth muscle contraction, inhibition of neuroblastoma cell differentiation, chemotaxis, tumor cell invasion, and antiproliferative effects on some cell types (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 2Moolenaar W.H. Kranenburg O. Postma F.R. Zondag C.M. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar). The intracellular biochemical signaling events that mediate the effects of LPA include stimulation of phospholipase C and increases in cytoplasmic calcium concentration, inhibition of adenylyl cyclase, and activation of phosphatidylinositol 3-kinase, the Ras-Raf-MAP kinase cascade, and Rho GTPases and Rho-dependent kinases (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 2Moolenaar W.H. Kranenburg O. Postma F.R. Zondag C.M. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar). The Ras-Raf-MAP kinase and Rho pathways stimulate the transcription factors ternary complex factor and serum response factor, respectively. Ternary complex factors and serum response factors synergistically activate transcription of immediate-early genes, such as c-fos, by binding to serum response element (SRE) in the promoters (7Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar). It has been demonstrated that specific G protein-coupled receptors (GPCRs) present on many types of cells mediate the cellular activities of LPA (2Moolenaar W.H. Kranenburg O. Postma F.R. Zondag C.M. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar, 8Durieux M.E. Lynch K.R. Trends Pharmacol. Sci. 1993; 14: 249-254Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 9Van der Bend R.L. Brunner J. Jalink K. van Corven E.J. Moolenaar W.H. van Blitterswijk W.J. EMBO J. 1992; 11: 2495-2501Crossref PubMed Scopus (178) Google Scholar). Several groups have just reported the isolation of cDNAs encoding two structurally different GPCRs for LPA. Hechtet al. isolated a mouse cDNA termed ventricular zone gene-1 (vzg-1) encoding one such GPCR. When overexpressed in neocortical cells, the vzg-1 receptor mediated LPA-induced morphological changes, inhibition of adenylyl cyclase, and increases in 3HLPA-binding sites (10Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar). Our laboratory identified human Edg2, a human homolog of vzg-1, as a functional receptor for LPA (11An S. Dickens M.A. Bleu T. Hallmark O.G. Goetzl E.J. Biochem. Biophys. Res. Commun. 1997; 231: 619-622Crossref PubMed Scopus (212) Google Scholar). Expression of Edg2 in HEK293 and Chinese hamster ovary cells led to elevated responses of LPA-induced SRE transcriptional reporter gene activation and increases in 3HLPA-binding sites. The amino acid sequences of mousevzg-1 and human Edg2 proteins are 96% identical, suggesting that they are the same LPA receptor in two different species. Guoet al. isolated a cDNA encoding a different GPCR fromXenopus oocytes (12Guo Z. Liliom K. Fischer D.J. Bathurst I.C. Tomei L.D. Kiefer M.C. Tigyi G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14367-14372Crossref PubMed Scopus (178) Google Scholar). Overexpression of this cDNA in oocytes potentiated oscillatory Cl− currents in response to LPA that could be attenuated by specific antisense oligonucleotides. This Xenopus LPA receptor shows little overall sequence similarity to the Vzg-1/Edg2 LPA receptors. Some functional studies have implied that multiple subtypes of LPA receptors with distinctive signaling properties mediate diverse cellular effects of LPA (13Liliom K. Bittman R. Swords B. Tigyi G. Mol. Pharmacol. 1996; 50: 616-623PubMed Google Scholar, 14Tigyi G. Dyer D.L. Miledi R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1012-1908Crossref Scopus (146) Google Scholar). To fully define the biological functions of LPA, we sought to identify novel subtypes of LPA receptors at a molecular level. In this study, we discovered a human cDNA termed edg4 encoding a GPCR with an amino acid sequence similar but not identical to human Edg2. We demonstrated that Edg4 is a novel subtype of LPA receptor functionally analogous to Edg2, but with a different tissue distribution. Oleoyl-LPA, lysophosphatidyl-choline, lysophosphatidyl-ethanolamine, lysophosphatidyl-serine, phosphatidic acid (PA), sphingosine 1-phosphate, fatty acid-free human serum albumin, and bovine serum albumin were purchased from Sigma. The mammalian expression vector pCDEF3 (15Goldman L.A. Curone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (151) Google Scholar), with a human elongation factor 1α promoter, was a generous gift from Dr. Jerome Langer (University of Medicine and Dentistry-New Jersey). Lipofectin and DMRIE-C lipofection reagents for transfection and OPTI-MEM medium were from Life Technologies, Inc. Jurkat leukemic T cells were obtained from Dr. Arthur Weiss (UCSF). Cell culture media and fetal bovine serum were from UCSF Cell Culture Facilities. Pertussis toxin (PTX) was purchased from CalBiochem (La Jolla, CA). Recombinant Clostridium botulinum C3 ADP-ribotransferase (C3 exoenzyme), which specifically ADP-ribosylates Rho (16Kumagai N. Morii N. Fujisawa K. Nemoto Y. Narumiya S. J. Biol. Chem. 1993; 268: 24535-24538Abstract Full Text PDF PubMed Google Scholar), was kindly provided by Drs. S. Narumiya and Y. Saito of Kyoto University (Kyoto, Japan). Human multiple tissue Northern blots were obtained fromCLONTECH (Palo Alto, CA). Plasmid pGL3-basic and luciferase assay reagents were from Promega (Madison, WI). 1-Oleoyl oleoyl-9,10-3HLPA with a specific activity of 56.2 Ci/mmol was from NEN Life Science Products. The BLASTN program was used to search dbEST division of GenBank™ for sequences homologous to that of human edg2 (11An S. Dickens M.A. Bleu T. Hallmark O.G. Goetzl E.J. Biochem. Biophys. Res. Commun. 1997; 231: 619-622Crossref PubMed Scopus (212) Google Scholar). A cDNA clone (GenBank™ accession number 755526) with its 5′ sequence (GenBank™ accession numberAA419064) similar but not identical to the 5′ region of edg2was identified and obtained from I.M.A.G.E. Consortium through Genome Systems (St. Louis, MO). The entire 1.7-kb insert of clone 755526 was sequenced on both strands using an ABI automated DNA sequencer (Howard Hughes Medical Institute DNA core facility, UCSF). The nucleotide sequence of 755526 was found to be highly homologous to that of humanedg2 and therefore was redesignated human edg4. The 1.7-kb insert was cut out by EcoRI and NotI and subcloned into the mammalian expression vector pCDEF3. The 1.1-kb cDNA of human edg2 coding region was cleaved from Edg2/RSV (11An S. Dickens M.A. Bleu T. Hallmark O.G. Goetzl E.J. Biochem. Biophys. Res. Commun. 1997; 231: 619-622Crossref PubMed Scopus (212) Google Scholar) and also subcloned into pCDEF3. The resulting expression constructs were designated Edg4/EF3 and Edg2/EF3, respectively. The construction of an SRE luciferase reporter gene plasmid containing four copies of SRE (5′-AGGATGTCCATATTAGGACATCT) and a TATA box has been previously described (11An S. Dickens M.A. Bleu T. Hallmark O.G. Goetzl E.J. Biochem. Biophys. Res. Commun. 1997; 231: 619-622Crossref PubMed Scopus (212) Google Scholar). Jurkat T cells were co-transfected with the SRE-luciferase reporter plasmid at a 1:10 ratio in combination with either Edg4/EF3, Edg2/EF3, or empty pCDEF3 vector using DMRIE-C reagent. After 4 h of transfection incubation in OPTI-MEM containing 10% fetal bovine serum, cells were washed and starved in serum-free RPMI 1640 at 37 °C for 8 h. Cells were then washed and resuspended in serum-free RPMI 1640, and aliquots of 1 × 105 cells were transferred into 96-well plates. LPA and other phospholipids dissolved in serum-free RPMI 1640 containing 0.1 mg/ml of human serum albumin were added to the cells followed by a 10-h incubation at 37 °C. Cells were then lysed by Reporter Lysis Buffer (Promega), and luciferase activities were measured using a Turner Designs 20/20 luminometer. To assess a G protein requirement, some aliquots of cells were incubated in the presence of 50 ng/ml of PTX, 10 μg/ml C3 exoenzyme, or both toxins during serum starvation and LPA treatment. Jurkat T cells (1 × 107/ml in OPTI-MEM) were transfected with 2 μg of Edg4/EF3 or empty pCDEF3 vector for 4 h at 37 °C using Lipofectin. The transfected cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum for 12 h at 37 °C and washed three times with phosphate-buffered saline before assessment of binding. Duplicate 0.2-ml aliquots of 2 × 106 cells were incubated with 200,000 cpm of 3HLPA in 0.25% bovine serum albumin-phosphate-buffered saline binding buffer for 45 min at 0 °C. The final concentration of 3HLPA in the binding incubations was 10 nm. The binding cell suspensions were passed through GF/C filters that were washed with 12 ml of ice-cold phosphate-buffered saline containing 0.05% Tween-20, air-dried, and added to scintillation fluid for quantification of radioactivity bound to the cells. Total and nonspecific binding were evaluated in the absence and the presence of 10 μmnonradioactive LPA, respectively. Specific binding was calculated from the difference in cpm between total binding and nonspecific binding. The 1.7-kb insert of Edg4/EF3 and the 1.1-kb insert of Edg2/EF3 were labeled with 32P and used as probes in Northern blot analyses. Northern blots containing 2 μg of poly(A)+ RNA from various human tissues and cancer cells in each lane were hybridized and washed under high stringency conditions (17Sambrook J. Fritsh F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.37-7.52Google Scholar). Blots were exposed to Kodak XAR film for 24 h at −70 °C with one intensifying screen. Among several sequences identified from a search of dbEST, one human cDNA sequence (GenBank™ accession number AA419064) was found to be highly similar but not identical to that of the humanedg2 cDNA in the 5′ region. The corresponding 1.7-kb cDNA clone (GenBank™ accession number 755526) contains the sequence of the full-length coding region of a novel protein that is 46% identical and 72% similar to the human Edg2 LPA receptor (Fig.1). We designated this cDNA as humanedg4 and its protein product as human Edg4 protein. The Edg4 protein consists of 382 amino acids with an estimated molecular weight of 42,626. It has some of the common features of a GPCR, including seven hydrophobic segments, potential N-linked glycosylation sites at the N terminus, and phosphorylation sites for serine/threonine kinases in the intracellular regions. Edg4 also possesses unique characteristics distinct from most other GPCRs. In Edg4, an alanine replaces a proline that is usually conserved in the NPXXY sequence of the seventh transmembrane domain. As in Edg2, the first extracellular loop of Edg4 lacks a cysteine residue that may form a disulfide bond with another cysteine in the second extracellular loop in most other GPCRs. The Edg4 protein is related not only to Edg2 but also to several “orphan” GPCRs, with 34% identity to rat H218 (18MacLennan A.J. Browe C.S. Gaskin A.A. Lado D.C. Shaw G. Mol. Cell. Neurosci. 1994; 5: 201-209Crossref PubMed Scopus (104) Google Scholar), 31% to human Edg1 (19Hla T. Maciag T. J. Biol. Chem. 1990; 265: 9308-9313Abstract Full Text PDF PubMed Google Scholar), and 30% to human Edg3 (GenBank™ accession number X83864). Recently, the human gene locus of edg4 was sequenced and located on chromosome 19p12 (GenBank™ accession number AC002306). Given the high degree of sequence homology of Edg4 protein with previously characterized LPA receptors Edg2 and Vzg-1, we predicted that Edg4 was a receptor for LPA or a structurally similar lipid mediator. Edg4 protein was thus overexpressed in Jurkat T cells for studies of LPA-induced responses and of radioactive LPA binding. Jurkat cells were chosen because they responses to LPA in the SRE reporter gene When co-transfected with the SRE-luciferase reporter Edg4, as Edg2, mediated increases in reporter gene expression by 1 LPA to The structurally related lysophosphatidyl-choline, lysophosphatidyl-ethanolamine, lysophosphatidyl-serine, and sphingosine at a concentration of 1 to increases in luciferase expression The Jurkat cells transfected with empty pCDEF3 vector in response to LPA or other phospholipids The activation of LPA-induced reporter gene in and Jurkat cells was at 1 LPA, a at LPA, and an of 10 the effect of LPA in both and Jurkat cells but with much of at The of and reporter activation of Edg4 was four times that of Edg2 2 and Pertussis toxin and botulinum C3 specifically and respectively. LPA-induced activation of the SRE reporter gene in both and Jurkat cells was partially by or C3 pretreatment The two toxins added inhibited the effects of LPA. that both of Gi and Rho are in from the Edg2 and Edg4 receptors to the SRE reporter gene 3HLPA binding is characterized by a nonspecific to its binding to cell Jurkat T cells, we were to nonspecific binding of 3HLPA with that of membrane The 3HLPA binding in the Jurkat cells was cpm In the same number of Jurkat cells, the specific binding was cpm which was the When calculated in of receptor Jurkat cells sites and Edg4 binding sites. Edg4 in Jurkat cells in increases in the number of specific binding sites for LPA. The other lysophosphatidyl-ethanolamine, and to 3HLPA binding to or Jurkat cells not The of mRNA encoding edg2 and edg4 in a of human tissues and cancer cells was by Northern blot The edg2 transcripts were found in human tissues with the in and the in and the edg2 transcripts also were in and but were in and cells In the two edg4 transcripts of 8 and 1.8 were not in human tissues as as and a of different from edg2 The was in and the was in the and the edg4 transcripts were in and edg2 transcripts were but were found in edg2 was In cancer cells, the of found in cell the was only in and cells were also In a of was in and cells The recent of the Vzg-1/Edg2 proteins as functional LPA receptors has allowed a sequence-based search for new genes that may encode novel LPA receptors. A human was identified by searching the dbEST and found to encode a GPCR that the human Edg2 LPA receptor SRE reporter gene assay demonstrated that Edg4 is a novel subtype of functional LPA receptor. with Edg2, Edg4 was in transcription in transfected Jurkat T cells 2 and In several difference in the of Edg2 and Edg4 mRNAs was in the transfected cells by Northern blot not because the cell expression of Edg2 and Edg4 receptor proteins were not measured in the study, the in activities could either that Edg4 protein was at a in cells or that Edg4 a receptor activity Edg2. Edg4 is an LPA receptor was also by increases in binding sites of 3HLPA to Jurkat cells. to high nonspecific binding of LPA to cell and the presence of binding in cells, binding of Edg4 and were not and Jurkat cells a number of specific binding sites to 3HLPA and a LPA-induced calcium response not they not to LPA in the SRE reporter gene assay 2 and This is to the presence of LPA receptors of a human of the receptor (12Guo Z. Liliom K. Fischer D.J. Bathurst I.C. Tomei L.D. Kiefer M.C. Tigyi G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14367-14372Crossref PubMed Scopus (178) Google Scholar) or an as subtype in Jurkat cells. has been in the activation of cellular and GTPases in some cell including Cell. 1996; PubMed Scopus (178) Google Scholar, R. J. Biol. Chem. 1997; PubMed Scopus Google Scholar). many of the reported effects of be to its is a of intracellular and extracellular signaling Cell. 1996; PubMed Scopus (178) Google Scholar, R. J. Biol. Chem. 1997; PubMed Scopus Google Scholar). Our demonstrated that activation of reporter gene was on Edg2 and Edg4 the of was high that of LPA. This that PA, on Edg2 and Edg4 is a much for receptors. different human tissue of edg4 and edg2 mRNAs was revealed by Northern blots In adult human and tissues mRNAs are edg4 mRNAs are In are edg4 mRNAs are the most The specific of containing edg4 mRNAs and the functions mediated by Edg4 in cells to be The tissue-specific expression of edg2 and to different cancer cell lines. The two transcripts of edg4 also and cancer expression The sequences and protein of the edg4 transcripts is that the cancer cells that edg2 and the of cells in cells in and not a of edg2 or the of edg4. The between the various edg2 and receptor and functions in cell and the gene expression of edg2 and edg4 is to be differently in different tissues and cells. LPA receptors in to at three distinct G and W.H. Kranenburg O. Postma F.R. Zondag C.M. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar). of phospholipase C and a of intracellular of Gi adenylyl and the to transcriptional activation mediated by ternary complex of the which to and transcriptional activation mediated by serum response The and pathways synergistically stimulate transcription of many genes containing SRE in promoters (7Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar). studies of Hechtet al. demonstrated that mouse Edg2 mediated inhibition of adenylyl a and cell morphological GTPases (10Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar). Our that and activation of transcription in Jurkat cells a Gi protein and C3 Rho We were to Edg2 and Edg4 also to which to an in of and intracellular calcium because were calcium responses to LPA in cells In we have discovered a human cDNA encoding a novel GPCR, Edg4, which has sequence similar to the previously identified human Edg2 LPA receptor. an reporter gene assay and a 3HLPA binding we have demonstrated that Edg4 is a novel functional receptor for LPA. Northern blot revealed that mRNAs for edg2 and edg4 have different tissue and cell distribution. The existence and distinctive tissue expression of structurally different LPA receptors may provide one basis for tissue-specific functions of LPA and permit independent regulation of each subtype of LPA receptor.